Tamoxifen induced multinucleated cells (symplasts) and distortion of seminiferous tubules in rat testis

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg·kg-1·d-1, 400 mg·kg-1·d-1 or 800 mg·kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.

Cycloheximide prevents production of arresting,a fraction of 30-50 kDa obtained from seminiferous tubule conditioned medium

Aim: To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermato-genesis and sperm count in male rats. Methods: The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined. Results: The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control. Conclusion: The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.

Stereological measurement of rat＇s seminiferous tubule

Background The morphological measurements of seminiferous tubules are important in the studies of testis tissues. The purpose of this study was to evaluate the feasibility of using a stereological method to measure the geometric parameters of seminiferous tubule and to optimize the method. Methods A stereological image processing program was developed with Delphi for the stereological measurement. Fields of view were obtained from 15 healthy Wistar rats＇ testis tissues （n=247）. The diameter, area and volume of seminiferous tubule were estimated with the image processing program by two individual observers. The area results were compared with those obtained by the standard morphometric method of planimetry, Results Diameter measurements showed the diameters of different seminiferous tubules were almost the same and the mean value of about 50 tubules could be a good representation of the whole structure. Area measurements indicated there was no significant difference between stereology and planimetry （P 〉0.05）. But the stereological method required about 45% less time. Volume measurement showed the inter-observer variability was small （P 〉0.05） and the reproducibility of the stereological method was good. Conclusion The stereological technique was practical and efficient in the quantitative measurement of the rat＇s seminiferous tubule.

Study on Transplanting Germ Like Cells Differentiated from Embryonic Stem Cell into Mouse Seminiferous Tubules

Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, stella, fragilis, Tex14, Sry, Hsp90-a, Stra8 and integrin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body （EB） derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1＋ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-a, Stra8 and integrin a6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.

Effects of Cypermethrin on Male Reproductive System in Adult Rats

Objective To evaluate effects of cypermethrin on the testis histology and testosterone, LH and FSH in adult male Sprague-Dawley rats. Methods The intact adult male rats were randomly divided into five groups and were treated with cypermethrin at doses of 0, 7.5, 15, 30, or 60 mg/kg per day by oral gavage for 15-days. After the treatments, serum was collected for hormone assays. The testes, epididymides, seminal vesicles, and prostates were excised and weighed. The right testis was frozen for daily sperm production and the left one was processed for histopathology. Results Daily sperm production decreased significantly in 30 and 60 mg/（kg.day） groups. Testicular structure abnormalities included atrophic and distorted seminiferous tubules, deformed and disordered arrangement of germ cells, reduced germ cells, Sertoli cells and Leydig cells, vacuolization and multinucleated formations of spermatids in the cypermethrin-treated rats. Vacuolization was found in Sertoli cells and the deformed nucleus was noted in Leydig cells. Serum testosterone reduced significantly in 30 and 60 mg/（kg.day） groups. Serum FSH increased significantly in 60 mg/（kg.day） group. Conclusion Cypermethrin induces impairments of the seminiferous tubules structure and spermatogenesis in the rats. The damages of the male reproductive system may be attributed to the imbalance of circulating testosterone.

Blood-testis barrier and spermatogenesis： lessons From genetically-modified mice

The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions （TJs）, adhesion junctions and gap junctions （GJs）. Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.

Effect of a single dose of malathion on spermatogenesisin mice

<abstract>Aim: To observe the acute effect of the organophosphorous insecticide malathion on testicular function in mice. Methods: The effects of a single dose of malathion [240 mg/kg (1/12 LD50)] on plasma acetylcholinesterase (ACE) activity, spermatozoa (epididymal cauda counts and teratozoospermia), testis and plasma testosterone concentration) were evaluated at day 1,8, 16, 35 and 40 after treatment. Results: The sperm count was decreased significantly 24 h after treatment and teratozoospermia was increased at day 35 and 40. The height of the seminiferous epithelium and the diameter of tubular lumen were decreased at day 8. The percentage of tubular blockade was increased between day 8 and 35. A decrease in testosterone plasma level was observed at day 16 after treatment. Conclusion: Malathion damages male reproduction. The depletion of seminiferous tubules and the increase in teratozoospermia may be a genotoxic damage to the renewing spermatogonia, but the possibility of spermatogenic/ spermiogenic disfunction due to a decrease in the plasma testosterone level can not be ruled out.

Contraceptive effect of Curcuma longa (L.) in male albino rat

Aim: To study the contraceptive effect of the crude extracts of Curcuma longa in male albino rats. Methods: Rats were fed orally with Curcuma longa aqueous and 70 % alcoholic extract for 60 days (500 mg·kg-1· day-1). Results: A reduction in sperm motility and density was observed in both the treated groups. Conclusion: Curcuma longa may have affected the androgen synthesis either by inhibiting the Leydig cell function or the hypo-thalamus pituitary axis and as a result, spermatogenesis is arrested.

Effect of ligustrum fruit extract on reproduction in experimental diabetic rats

Aim: To study the effect of ligustrum fruit on spermatogenesis and blood gonadal hormones in diabetic rats.Methods: Experimental diabetes was induced in male Wistar rats with streptozotocin. Ligustrum fruit extract wasgiven by gastric gavage at a dose of crude drug 30 g·kg^(-1)·d^(-1) for 110 days. The serum gonadadotropic hormones andtestosterone were determined on d 60 and testicular histology examined on d 110. Results: In the control diabeticrats, the seminiferous tubules were dilated and the spermatogenic cells irregularly arranged. Spermatogenesis was arrest-ed with the number of spermatids highly reduced and spermatozoa not observed. In the treated rats, all types of sper-matogenic cells were practically normal. The serum luteinizing hormone (LH), follicle-stimulating hormone (FSH)and testosterone levels were higher in the treated than in the control rats, but the difference was insignificant. Conclu-sion: In experimental diabetic rats, ligustrum fruit extract protects the damaging effect of experimental diabetes onspermatogenesis. (Asian J Androl 2001 Mar; 3 : 71-73)

Normal and varicocele testis in adolescents

The authors reviewed the results of their research on the structure and composition of normal and varicocele semi-niferous tubules in adolescents. They give new evidences of normal structure of adolescent testis and demonstrate, forthe first time, the ultrastructural and immunohistochemical modifications of the lamina propria and basal lamina in theadolescent varicocele patients, which are similar to those observed in adults, but less severe, and of the adherence junc-tions in seminiferous tubules. They also report the presence of oxidative stress in adolescents limited to testis and notgeneralised as in the adults. These data are well correlated to different clinical studies that support the hypothesis of aprogressive course of varicocele and the need for surgical treatment in adolescent varicocele patients. (Asian J Androl2001 Dec; 3: 259-262)

Effect of lead chloride on spermatogenesis and sperm parameters in mice

Aim: To evaluate the effect of acute lead chloride exposure on testis and sperm parameters in mice. Methods: PbCl2, 74 mg/kg, was daily administered to sexually mature male mice for 3 days and the effects on the testicular histology and ultrastructure as well as the motility and density of spermatozoa in cauda epididymis were observed. An additional group of mice were treated for 1-3 days and were allowed to recover for 32 days to determine the reversibility of lead-induced changes. Results: The testicular weight, seminiferous tubular diameter and sperm counts were significantly decreased following 3 days of PbCl2 treatment, but were unaffected by shorter-term exposures. The changes caused by lead are mostly reversible. Conclusion: Acute lead chloride exposure injures the fertility parameters of male mice and the effects are partially reversible.

Production of Transgenic Animals Using Spermatogonial Stem Cells

Spermatogonial stem cells （SSCs） are a type of adult stem cell found in male mammals.These cells have the capacity for self renewal and are capable of differentiating in the niche of testis.They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life,transferring genetic information to the offspring.Since a technique for transplanting SSCs was first described by Brinster and his colleagues in 1994,more and more researchers have become interested in exploring the possibility of utilizing adult SSCs to generate transgenic animals.In this mini-review,we attempt to summarize the current research progress in the area of spermatogonial stem cells including the source,types and differentiation of the SSCs,and the application on transgenic animals,with a particular focus on the strategy of SSCs delivery including seminiferous tubule injection and spermatogonial stem cell transplantation.

Effect of tamoxifen on spermatogenesis and tubular morphology in rats

Aim: To observe the effect of tamoxifen citrate on spermatogenesis and tubular morphology in rats. Methods: The effect of tamoxifen citrate i.g. at doses of 400 and 800 mg·kg·day-1 in 0.1 mL olive oil for 30 days on seminiferous tubular morphology, seminiferous epithelial diameter (STD), epithelial height (SEH), epididymal sperm count and percent abnormal sperm were evaluated at day 1, 12 and 36 after treatment. Controls were given the vehicle. Results: The higher dose resulted in tubular atrophy on day 31. The STD, SEH and sperm count were decreased and the abnormal spermatozoa increased in a dose-dependent manner with the maximal effect on day 36. Conclusion: Tamoxifen citrate induces tubular shrinkage and atrophy and sperm abnormality at a dose-dependent manner.

Ectopic porcine spermatogenesis in murine subcutis： tissue grafting versus cell-injection methods

Fragments of testis tissue from immature animals grow subcutaneous areas of immunodeficient mice. The same results testes of rodents are injected into the subcutis of nude mice. and develop spermatogenesis when grafted onto are obtained when dissociated cells from immature Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient （SCID） and NOD/Shi- SCID, IL-2Rγc^null （NOG） mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell- injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not signifcant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.

Potential of triptolide in reproductive management of the house rat,Rattus rattus

Mature and healthy male house rats,Rattus rattus(n=160)were fed on bait(cracked wheat:powdered sugar,98:2)containing different concentrations of triptolide(0.1,0.05,0.025 and 0%)for 7 and 14 days in no-choice and bi-choice feeding tests in the laboratory.The objective of the study was to record the antifertility affects of triptolide after 30 and 60 days of termination of treatment.Results revealed no significant effect of triptolide treatment on weights of testis,epididymis,seminal vesicles and prostate gland of rats.Overall,sperm motility,live sperm count,sperm density and sperm morphology in the cauda epididymal fluid were found to differ significantly(P≤0.05)between untreated and treated groups of rats.The major effect of triptolide on sperm morphology was in the form of sperm head tail separation,which was up to 56.0%in rats treated for 14 days in no-choice and autopsied after 30 days.A significant effect(P≤0.05)of triptolide treatment was observed on the histomorphology of the testis,which included a dose-dependent decrease in diameter of seminiferous tubules,thickness of germinal epithelium and numbers of various spermatogenic cells.Cell associations in the seminiferous epithelial cycle were poorly developed in rats ingesting medium(4.7-5.1 mg/100 g bw)and high doses(6.9-7.2 mg/100 g bw)of triptolide than rats ingesting low doses(1.8-2.3 mg/100 g bw)and untreated rats.The cell stages affected had not recovered fully within the 60 day period following triptolide withdrawal.The present study suggests the potential of triptolide in reproductive management of Rattus rattus.

Effect of Hydro-ethanolic (1:1) Extract of Stephania Hernandifolia and Achyranthes Aspera in Composite Manner on Testicular Activity in Male Albino Rat:A Dose Dependent Analysis

Objective To investigate the effect of hydro-ethanolic extraet of Stephania hernandifolia leaves and Aehyranthes aspera roots in composite manner at the ratio of 1：3 on testicular activity in male rats. Methods Rats were divided into 4 groups with 8 animals in each group. The control （group A） received 0. 5 ml of olive oil/100 g body weight orally, other three groups were treated with said extract orally at a dose of 0.4 mg/g （group B） or 0.8 mg/g （group C） or 1.6 mg/g body weight （group D） respectively for 28 d. On 29th day of experiment, the animals were sacrificed. Sperm concentrations in cauda epididymis and biochemical markers like testicular cholesterol, androgenic key enzyme activity, plasma testosterone level seminal fructose level, glutamate oxaloacetate transaminase （GOT） and glutamate pyruvate transaminase （GPT） activities and serum triglyceride levels were measured following standard methods. ResuIts Extract treated animals in all doses resulted in a significant （P〈O. 05）decrease in sperm concentration, testicular androgenic key enzyme activity, plasma testosterone and seminal vesicle fructose levels along with an increase in testieular cholesterol level Animals treated at a dose of O. 8 mg/g body weight showed more promising result without causing any metabolic toxicity compared with other doses. Histological study also supported the biochemical results. The minimum but most effective dose i.e. 0.8 mg/g body weight had an inhibitory effect on implantation focused here by mating experiment.Conclusion The composite efficacy as male contraceptive extract of S. hernandifolia and A. aspera has a potent that may provide clues to the pharmaceutical industries for male contraceptive development